This invention relates to a calcium-phosphate type hydroxyapatite for chromatographic separation and a process for producing the same.
More particularly, the invention is concerned with a novel calcium-phosphate type hydroxyapatite having specific crystal properties and useful as a column packing material for separation of biopolymers.
The term apatite-structure compounds is generically used to denote a large group of compounds having a common formula M.sub.10 (ZO.sub.4).sub.6.X.sub.2 where M represents a metallic atom of Ca, Ba, Sn, Mg, Na, K, Pb, Cd, Zn, Ni, Fe, Al or the like, ZO.sub.4 represents an acid radical, such as PO.sub.4, AsO.sub.4, VO.sub.4, SO.sub.4, or SiO.sub.4, and X represents an OH or F atom (group). The present invention is directed to a calcium phosphate compound of the above general formula in which M is essentially Ca, ZO.sub.4 essentially PO.sub.4, and X essentially OH.
The calcium-phosphate type hydroxyapatite, Ca.sub.10 (PO.sub.4).sub.6 (OH).sub.2 (hereinafter termed "hydroxyapatite" unless otherwise noted), has attracted growing attraction in recent years as a material for biomedical use, typically for artificial teeth and bones. This is because it closely resembles in composition to the inorganic ingredients of the teeth and bones of vertebrate animals and exhibits good affinity for living bodies. The affinity for living organisms has rendered it useful as a packing material to be filled in columns for chromatographic separation of biopolymers, for example, proteins, enzymes, and nucleic acids.
Conventionally, such hydroxyapatites are synthesized in most cases by
(1) wet synthesis involving an aqueous solution reaction in which a water-soluble calcium salt and phosphate are allowed to react in aqueous solution,
(2) dry synthesis involving a high-temperature solid-phase reaction in which calcium phosphate and calcium carbonate are allowed to react in the presence of water vapor at 900.degree. to 1400.degree. C., or
(3) hydrothermal synthesis involving a reaction under high-temperature, high-pressure steam in which calcium hydrogen-phosphate is hydrolyzed, for example, at 200.degree. C. and at 15 atm.
In addition to these, a new synthetic process has been proposed, for example, by Patent Application Publication No. 500447/1980.
The hydroxyapatites obtained in the foregoing processes have been in the form of plates which have to be finely divided particularly when used as column packing material for chromatographic separation.
The plates are thus divided into tiny pieces varied in shape and size. The irregular pieces of hydroxyapatite cannot be packed uniformly or densely in the column for chromatographic separation. The packing material so obtained can hardly be considered satisfactory in performance, both in separability and selectivity.
In an effort to overcome the difficulties of the conventional hydroxyapatites as column packing materials for chromatographic separation, we have made extensive studies and experiments. As a result, it has now been found that, when such a hydroxyapatite is employed as a packing material for chromatographic separation, the size and shape of the finely divided product constitute very important factors and that, in particular, the shape should be spherical and the size distribution be within a narrow range, and further that the hydroxyapatite in the form of spherulites having a mean particle diameter of 0.5 to 50 .mu.m, preferably 1 to 10 .mu.m, achieves adequate separability and selectivity.
The term "particle diameter" as used herein refers to the diameter of the apatite "crystallite aggregate". This differs from the "crystallite size" of the apatite.
The term "crystallite" means a micro-crystal. As the crystallite grows in size, the phrase "grain size" is substituted for the phrase "crystallite size" in the industry.
While the hydroxyapatite prepared according to the present invention is in the form of spherulites having a mean "particle diameter" of 0.5 to 50 .mu.m, the "crystallite size" of the present apatite ranges from between 0.01 to 0.05 .mu.m (i.e., from 100 to 500 .ANG.) wide and from 0.05 to 0.3 .mu.m (i.e., from 500 to 3,000 .ANG.) long.
The present invention is based upon these newly found facts.
Therefore, the object of the invention is, in view of the fact that the hydroxyapatites proposed thus far have had inadequate separability and selectivity when used as column packing materials for chromatographic separation of biopolymers, to provide a novel calcium-phosphate type apatite for chromatographic separation having unique shape and size and excels in separability and selectivity.